Advanced optical microscopy techniques offer unique opportunities to investigate biological processes in vivo. The ability to image tissues or organisms in three dimensions (3D) and/or over time (4D imaging) permits a wide range of applications in neuroscience, immunology, cancer research, and developmental biology. (See, e.g., Mertz, Curr. Opin. Neurobiol. 14, 610-616, (2004); Kerr, J. N. D. & Denk, W., Nature Reviews Neuroscience 9, 195-205, (2008); Friedl, P., Current Opinion in Immunology 16, 389-393, (2004); Bousso, P., Current Opinion in Immunology 16, 400-405, (2004); Provenzano, P. P., et al., Trends in Cell Biology 19, 638-648, (2009); Keller, P. J., et al., Science 322, 1065-1069 (2008); McMahon, A., et al., Science 322, 1546-1550 (2008); and Mavrakis, M., et al., Development 137, 373-387, (2010), the disclosures of each of which are incorporated herein by reference.) Fundamental light-matter interactions, such as light scattering, absorption, and photo-induced biological toxicity, set the limits on the performance parameters of various imaging technologies in terms of spatial resolution, acquisition speed, and depth penetration (how deep into a sample useful information can be collected). Often, maximizing performance in any one of these parameters necessarily means degrading performance in the others. (See, e.g., Ji, N., et al., Curr. Opin. Neurobiol. 18, 605-616, (2008) and Vermot, J., et al., HFSP Journal 2, 143-155 (2008), the disclosures of each of which are incorporated herein by reference.)
Such tradeoffs in performance are seen in comparing two current well-known 4D fluorescence imaging techniques of raster point scanning (RAPS) microscopy and light sheet (LISH) microscopy: RAPS excels in imaging of flat samples, while LISH excels in imaging of 3D samples and allows higher acquisition speed and lower phototoxicity. In RAPS microscopy, also known in the literature as laser scanning microscopy (LSM), the images are generated one voxel at a time by raster-scanning a tightly-focused laser spot through the sample, and 3D resolution is achieved by spatial-filtering of the emitted signal or by relying on nonlinear excitation to produce signal only at the focus spot (See, e.g., Pawley, Handbook of Confocal Microscopy, 3rd Edition, New York: Springer (2006), the disclosure of which is incorporated herein by reference.) The acquisition speed of RAPS is, however, limited since the image is collected one voxel at a time. Also, the phototoxicity quality of RAPS is relatively high due to the high laser light intensity concentrated at the focus spot.
LISH microscopy is a century-old technology that has seen much development and refinement in recent years, under names ranging from Orthogonal Plane Fluorescence Optical Sectioning (OPFOS), Thin Laser light Sheet Microscopy (TLSM), Selective Plane Illumination Microscopy (SPIM) (FIG. 1A, high-speed imaging of live zebrafish heart), Objective Coupled Planar Illumination (OCPI) (FIG. 1B, high-speed calcium imaging of neurons), ultramicroscopy (FIG. 1C, blood vessel system of mouse embryo), and Digital Scanned Laser Light Sheet Fluorescence Microscopy (DSLM) (FIG. 1D, in Coto imaging of developing zebrafish embryo), among others. (See, e.g., Siedentopf, H. & Zsigmondy, R., Ann. Phys.-Berlin 10, 1-39 (1902); Voie, A. H., et al., J. Microsc.-Oxf. 170, 229-236 (1993); Fuchs, E., et al., Opt. Express 10, 145-154 (2002); Huisken, J., et al., Science 305, 1007-1009 (2004); Holekamp, T. F., et al., Neuron 57, 661-672 (2008); Dodt, H. U. et al., Nat. Methods 4, 331-336 (2007); Huisken, J. & Stainier, D. Y. R., Development 136, 1963-1975 (2009); and Keller, P. J. & Stelzer, E. H. K., Curr. Opin. Neurobiol. 18, 624-632 (2009), the disclosures of each of which are incorporated herein by reference.)
In LISH, (FIG. 1E) a planar sheet of light is used to illuminate the sample, generating fluorescence signal over a thin optical section of the sample, which is then imaged from the direction orthogonal to the light sheet, with a wide-field imaging camera. Axial sectioning results from the thinness of the light sheet, while lateral resolution is determined by the detection optics. The orthogonal geometry between the illumination and detection pathways of LISH, compared to the collinear geometry of conventional microscopes, not only enables higher imaging speed due to the parallel image collection (millions of voxels can be imaged simultaneously), but also reduces phototoxicity because only a single focal plane of the sample is illuminated at a time, and because the laser light power is spread out in space over the extended sheet (as compared a single focus spot in the RAPS case). The low photototoxicity of LISH makes it the ideal choice for long-term time-lapsed imaging where a live biological sample is observed over an extended time window, from several hours to several days. LISH microscopy also allows imaging of a 3D sample from different views, thus facilitating improved coverage and resolution for samples such as a whole developing embryo. LISH, however, requires that the sample can be optically accessed from the side, which precludes samples that are flat.
Thus, it can be seen that the two imaging modalities of RAPS and LISH microscopy are complementary, each ideally suited for a particular type of samples. Ideally, a biomedical research laboratory would like to have access to both types of microscopy, allowing access to the widest possible selection of samples. However, there is a lack of commercially available LISH microscopes, and even if a commercial LISH microscope is available there is the high cost, both in monetary and space-related terms, associated with owning two different microscopes in order to do both LISH and RAPS microscopy.
Accordingly, it would be advantageous to develop an optical microscope that allows for the simultaneous performance of RAPS and LISH capable of providing new imaging capabilities heretofore unobtainable with conventional microscopy techniques.